Aims: To construct a genetically modified strain of Streptococcus mutans for dental caries prevention. The strain has significantly reduced cariogenicity owing to a deletion of the entire open reading frame for lactate dehydrogenase, and has excellent colonization potential through the production of a natural antibiotic called mutacin 1140. For use in human clinical trials, additional mutations were introduced to enable rapid elimination of the strain in case of adverse side effects and to increase genetic stability. Methods: Deletion mutations were introduced into the dal gene for D-alanine biosynthesis and the comE gene for genetic transformation. The resulting strain, A2JM, was tested for dependence on exogenous D-alanine and its ability to be eradicated from colonized rats. The strain was also tested for its ability to exchange DNA with another strain of S. mutans in in vitro and in vivo models. Conclusions: A2JM was completely dependent on exogenous D-alanine, but could colonize the oral cavity of rats in low numbers in the absence of dietary D-alanine. Results indicated that A2JM can scavenge D-alanine from other plaque bacteria. Lowering of the total oral bacterial load through daily application of chlorhexidine enabled virtually complete eradication of A2JM. The introduction of the comE gene did not significantly decrease the transformability of A2JM in in vitro or in vivo models. The addition of a deletion in the comE gene does, nonetheless, provide additional safety as it has a very low reversion frequency. Significance and Impact of the Study: Based on the safety and efficacy profiles established in vitro and in animal models, A2JM appears suitable for safe use in human clinical trials.